The broad, long-term objective of this program is to study inositol metabolism in humans. Inositol is a key cellular metabolite. It is the precursor for the synthesis of phosphatidylinositol, an essential membrane lipid, an anchor for proteins, and a core component of the recently elucidated signal transduction mechanisms. The level of inositol biosynthesis appears to be tissue-specific, affected by hormones and some diseases. Aberrant concentrations of inositol have been implicated in neuropathy, vascular abnormalities and development of cataracts associated with diabetes. The aim of the project is the molecular genetic analysis of the regulation of inositol metabolism. Specifically, the project proposed is the isolation of a cDNA clone encoding the human inositol-1-phosphate synthase and the establishment of a eukaryotic model system. The initial approach to cloning this gene will be the complementation of a yeast mutant strain, defective in the enzyme, with a clone bank of human cDNA in a yeast expression vector. Three alternate approaches which could be employed to isolate this clone are: 1) hybridization of the functionally analogous genes to clone banks of human cDNA; 2) polymerase chain reaction suing human mRNA as template and conserved regions of the fungal genes as primers, and; 3) purification of the human protein and subsequent generation of sequence information to synthesize precise probes for hybridization to clone banks. To establish a eukaryotic model system of inositol metabolism (biosynthesis and catabolism), it was essential to determine which organisms catabolized inositol. Cryptococcus neoformans can grow on inositol as a sole carbon source. In contrast, inositol does not support the growth of strains of S. cerevisiae and C. albicans. Preliminary results indicate that inositol biosynthesis also occurs in C. neoformans. Hence, C. neoformans is being examined as a potential model organism. An ideal model system to study inositol metabolism should have enzymic reactions similar to those found in higher eukaryotes. The initial biochemical, genetic, and molecular biological studies will be performed to determine the inositol biosynthetic and catabolic pathways of C. neoformans. A direct consequence of this propose research program will be attracting students to the biomedical sciences, providing a modern research environment and training in molecular genetics and encouraging future graduate studies. This project is ideal in that it requires the use of many basic molecular biological and genetic techniques.